Precise Synthesis of Organic Cocrystal Alloys with Full-Spectrum Emission Characteristics for the Stepless Color Changing Display.

Organic luminescent materials are indispensable in optoelectronic displays and solid-state luminescence applications. Compared with single-component, multi-component crystalline materials can improve optoelectronic characteristics. This work forms a series of full-spectrum tunable luminescent charge-transfer (CT) cocrystals ranging from 400 to 800 nm through intermolecular collaborative self-assembly. What is even more interesting is that o-TCP-Cor(x)-Pe(1-x), p-TCP-Cor(x)-Pe(1-x), and o-TCP-AN(x)-TP(1-x) alloys are prepared based on cocrystals by doping strategies, which correspondingly achieve the stepless color change from blue (CIE [0.22, 0.44]) to green (CIE [0.16, 0.14]), from green (CIE [0.27, 0.56]) to orange (CIE [0.58, 0.42]), from yellow (CIE [0.40, 0.57]) to red (CIE [0.65, 0.35]). The work provides an efficient method for precisely synthesizing new luminescent organic semiconductor materials and lays a solid foundation for developing advanced organic solid-state displays.

Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

Oriented Self-Assembly of Hierarchical Branch Organic Crystals for Asymmetric Photonics.

Organic hierarchical branch micro/nanostructures constituted by single crystals with inherent multichannel characteristics exhibit superior potential in regulating photon transmission for photonic circuits. However, organic branch micro/nanostructures with precise branch positions are extremely difficult to achieve due to the randomness of the nucleation process. Herein, by taking advantage of the dislocation stress field-impurity interaction that solute molecules deposit preferentially along the dislocation line, twinning deformation was introduced into microcrystals to induce oriented nucleation sites, and ultimately organic branch microstructures with controllable branch sites were fabricated. The growth mechanism of these controllable single crystals with an angle of 140° between trunk and branch is attributed to the low lattice mismatching ratio (η) of 4.8%. These as-prepared hierarchical branch single crystals with asymmetrical optical waveguide characteristics have been demonstrated as an optical logic gate with multiple input/out channels, which provides a route to command the nucleation sites and offers potential applications in the organic optoelectronics at the micro/nanoscale.

Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy.

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

Electrochemical Production of Glycolate Fuelled By Polyethylene Terephthalate Plastics with Improved Techno-Economics.

Electrochemical valorization of polyethylene terephthalate (PET) waste streams into commodity chemicals offers a potentially sustainable route for creating a circular plastic economy. However, PET wastes upcycling into valuable C2 product remains a huge challenge by the lack of an electrocatalyst that can steer the oxidation economically and selectively. Here, it is reported a catalyst comprising Pt nanoparticles hybridized with γ-NiOOH nanosheets supported on Ni foam (Pt/γ-NiOOH/NF) that favors electrochemical transformation of real-word PET hydrolysate into glycolate with high Faradaic efficiency (> 90%) and selectivity (> 90%) across wide reactant (ethylene glycol, EG) concentration ranges under a marginal applied voltage of 0.55 V, which can be paired with cathodic hydrogen production. Computational studies combined with experimental characterizations elucidate that the Pt/γ-NiOOH interface with substantial charge accumulation gives rise to an optimized adsorption energy of EG and a decreased energy barrier of potential determining step. A techno-economic analysis demonstrates that, with the nearly same amount of resource investment, the electroreforming strategy towards glycolate production can raise revenue by up to 2.2 times relative to conventional chemical process. This work may thus serve as a framework for PET wastes valorization process with net-zero carbon footprint and high economic viability.

Tracking the Replication-Competent Zika Virus with Tetracysteine-Tagged Capsid Protein in Living Cells.

Real-time imaging of viruses in living cells considerably facilitates the study of virus-host interactions. However, generating a fluorescently labeled recombinant virus is challenging, especially for Zika virus (ZIKV), which causes microcephaly in neonates. The monocistronic nature of the ZIKV genome represents a major challenge for generating a replication-competent genetically engineered ZIKV suitable for real-time imaging. Here, we generated a fluorescent ZIKV by introducing the biarsenical tetracysteine (TC) tag system. After separately inserting the TC tag at six sites in the capsid protein, we found that only when we inserted the TC tag at the site of amino acids 27/28 (AA27/28, or TC27) could the genetically engineered ZIKV be rescued. Importantly, the TC27 ZIKV is characterized as replication and infection competent. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the dynamic nuclear import behavior of the capsid protein. In addition, using the single-particle tracking technology, we acquired real-time imaging evidence that ZIKV moved along the cellular filopodia and entered into the cytoplasm via endocytosis. Thus, we provide a feasible strategy to generate a replication-competent TC-tagged ZIKV for real-time imaging, which should greatly facilitate the study of ZIKV-host interactions in living cells. IMPORTANCE Zika virus (ZIKV) is the mosquito-borne enveloped flavivirus that causes microcephaly in neonates. While real-time imaging plays a critical role in dissecting viral biology, no fluorescent, genetically engineered ZIKV for single-particle tracking is currently available. Here, we generated a replication-competent genetically engineered ZIKV by introducing the tetracysteine (TC) tag into its capsid protein. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the nuclear import behavior of the capsid protein and the endocytosis process of single ZIKV particle. Taken together, these results demonstrate a fluorescent labeling strategy to track the ZIKV-host interactions at both the protein level and the viral particle level. Our replication-competent TC27 ZIKV should open an avenue to study the ZIKV-host interactions and may provide applications for antiviral screening.

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Hierarchical Integration of Organic Core/Shell Microwires for Advanced Photonics.

The combination of multiple components or structures into integrated micro/nanostructures for practical application has been pursued for many years. Herein, a series of hierarchical organic microwires with branch, core/shell (C/S), and branch C/S structures are successfully constructed based on organic charge transfer (CT) cocrystals with structural similarity and physicochemical tunability. By regulating the intermolecular CT interaction, single microwires and branch microstructures can be integrated into the C/S and branch C/S structures, respectively. Significantly, the integrated branch C/S microwires, with multicolor waveguide behavior and branch structure multichannel waveguide output characteristics, can function as an optical logic gate with multiple encoding features. This work provides useful insights for creating completely new types of organic microstructures for integrated optoelectronics.

Single-Particle Tracking Reveals the Interplay between HIV-1 Reverse Transcription and Uncoating.

Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.

Synthesis of Dual-Emitting CdZnSe/Mn:ZnS Quantum Dots for Sensing the pH Change in Live Cells.

Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.

Construction of ratiometric Si-Mn:ZnSe nanoparticles for the immunoassay of SARS-CoV-2 spike protein.

The continuing global spread of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, has led to an unprecedented global health crisis. Effective and affordable methods are needed to diagnose SARS-CoV-2 infection. In this work, a ratiometric fluorescence probe, Si-Mn:ZnSe nanoparticles, was constructed through the electrostatic interaction between Si dots and Mn:ZnSe QDs, and the fluorescence of Mn:ZnSe QDs has a specifical response to H2O2. An immunocomplex was formed by the recognition of capture antibody/spike (S) protein/spike neutralizing antibody/biotinylated second antibody/streptavidin/biotinylated catalase (CAT). In the presence of S protein, CAT effectively catalyzed the decomposition of H2O2 in the system, and the fluorescence of Mn:ZnSe QDs was not specifically quenched. Based on this principle, a ratiometric immunoassay of SARS-CoV-2 S protein was established. The sensitivity of the proposed ELISA method was comparable to that of the commercial kit. In addition, this method can effectively distinguish the pseudo-SARS-CoV-2 virus and other pseudovirus. Therefore, this method provided a reliable and potential direction for diagnosing SARS-CoV-2 infection.

Design and Growth of Branched Organic Crystals: Recent Advances and Future Applications.

The rapid development of information technology has resulted in a growing demand for low-dimensional photonic materials. Organic semiconductor materials play an important role in various photonic devices due to their adjustable physicochemical properties, while individual organic crystals do not exhibit the desired performance due to the limitations of their simple structure. Branched organic crystals with inherent multichannel characteristics based on π-conjugated molecules are favorable components in optoelectronics. However, the preparation of branched organic crystals still faces great challenges before they can be applied in integrated optoelectronic devices. In this Review, the development and representative examples of branched organic crystals in terms of molecular design, synthesis, and advanced applications are discussed. We also provide a summary and outlook for the direction of future research on branched organic crystals as excellent candidates in photonic integrated circuits.

Novel Method of Clickable Quantum Dot Construction for Bioorthogonal Labeling.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

Magnetic bead-enzyme assemble for triple-parameter telomerase detection at single-cell level.

In this work, we developed a triple-parameter strategy for the detection of telomerase activity from cancer cells and urine samples. This strategy was developed based on magnetic bead-enzyme hybrids combined with fluorescence analysis, colorimetric assay, or adenosine triphosphate (ATP) meter as readout. The application of magnetic bead-enzyme hybrids has the advantages of magnetic separation and signal amplification. These detection methods can be used individually or in combination to achieve the optimal sensing performance and make the results more convincing. Among them, the ATP meter with portable size had easy operation and low cost, and this response strategy provided a higher sensitivity at the single-cell level. The designed strategy was suitable as naked-eye sensor and point-of-care testing (POCT) for rapid assaying of telomerase activity. Graphical abstract Magnetic bead-enzyme assemble for triple-parameter telomerase detection.

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

Live Visualization of HIV-1 Proviral DNA Using a Dual-Color-Labeled CRISPR System.

HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mapped based on the colocalized signals of QDs in living cells. Compared to the existing zinc finger proteins and TALENs, the CRISPR system is much easier to operate and more efficient in imaging of internal genomic loci. Therefore, the proposed method could be not only a powerful tool for imaging proviral HIV-1, but also a versatile platform to image single genomic loci in living cells.

Delaying Photobleaching of a Light-Switch Complex for Real-Time Imaging of Single Viral Particle Uncoating.

Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.

Basal CD38/cyclic ADP-ribose-dependent signaling mediates ATP release and survival of microglia by modulating connexin 43 hemichannels.

It is necessary to investigate the mechanisms underlying ATP release from neural cells, because extracellular ATP plays multiple important biological roles in the brain. CD38 is an ectoenzyme that consumes NAD(+) to produce cyclic ADP-ribose (cADPR), a potent agonist of ryanodine receptors. Our previous study showed that CD38 reductions led to microglial apoptosis. In this study, we used both murine microglial BV2 cells and primary microglial cultures as cellular models to test our hypothesis that basal CD38/cyclic ADP-ribose (CD38/cADPR)-dependent signaling plays a key role in ATP release, which mediates basal survival of microglia. We found that inhibition of CD38/cADPR-dependent signaling by CD38 silencing or 8-Bromo-cADPR, a ryanodine receptor antagonist, produced significant ATP release from BV2 microglia. Cx43 small interfering RNA and Cx43 hemichannel blocker 18-α-glycyrrhetinic acid completely prevented the CD38 silencing or 8-Bromo-cADPR-induced ATP release. Prevention of the ATP release could also be due to P2X7 receptor antagonists. Our study has further suggested a key role of ATP release in the microglial apoptosis induced by decreased CD38/cADPR-dependent signaling. In addition, by using primary microglial cultures, we found that 8-Bromo-cADPR also induced significant ATP release, which could be attenuated by 18-α-glycyrrhetinic acid. 8-Bromo-cADPR was also found to induce death of primary microglial cultures. In conclusion, our results have suggested novel roles of basal activation of CD38/cADPR-dependent signaling in mediating microglial functions and survival: It mediates ATP release from microglia by modulating Cx43 hemichannels, which can significantly affect microglial survival.

Design and Application of Biosafe Coronavirus Engineering Systems without Virulence.

In the last twenty years, three deadly zoonotic coronaviruses (CoVs)-namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2-have emerged. They are considered highly pathogenic for humans, particularly SARS-CoV-2, which caused the 2019 CoV disease pandemic (COVID-19), endangering the lives and health of people globally and causing unpredictable economic losses. Experiments on wild-type viruses require biosafety level 3 or 4 laboratories (BSL-3 or BSL-4), which significantly hinders basic virological research. Therefore, the development of various biosafe CoV systems without virulence is urgently needed to meet the requirements of different research fields, such as antiviral and vaccine evaluation. This review aimed to comprehensively summarize the biosafety of CoV engineering systems. These systems combine virological foundations with synthetic genomics techniques, enabling the development of efficient tools for attenuated or non-virulent vaccines, the screening of antiviral drugs, and the investigation of the pathogenic mechanisms of novel microorganisms.